The present disclosure relates in general to analytical instruments and methods for electrochemical testing. Specifically, the present disclosure relates to electrochemical detection of proteolysis of proteinaceous matrices. More specifically, new apparatus and methods are provided for detecting proteolysis of proteinaceous matrices including fibrin clots, extracellular matrix and similar collagen or related protein-based matrices, whether endogenous or synthetic.
Protein matrices are important for tissue function, tissue regeneration, wound healing, and hemostasis, among other things. For example many eukaryotic cells are enveloped by an extracellular matrix of proteins that provide structural support, cell and tissue identity, and autocrine, paracrine and juxtacrine properties for the cell and the matrix is thus required for normal tissue functions. In wound healing a cascade of molecular and cellular events initially lead to homeostasis, the prevention of blood loss. Fibrin plays a crucial role in hemostasis and wound healing as it forms a crosslinked proteinaceous matrix (clot) by means of a complex cascade of reactions with the final steps being the conversion of monomeric fibrinogen by thrombin, to form a crosslinked fibrin polymer, which is often referred to as a clot.
In addition to its role in hemostasis fibrin formation is common in a number of pathological and inflammatory conditions. For example, fibrin deposition (thrombosis) is associated with atherosclerosis, rheumatoid arthritis, glomerulonephritis, systemic lupus erythematosis, myocardial infarcts, pulmonary embolism, deep vein thrombosis, autoimmune neuropathies, granulomatous disease, parasitic infections and allograft rejection. There is also evidence that thrombosis plays a role in neurodegenerative disease. Similarly, collagenases play significant role in invasive cancer where they degrade the extracellular matrix in healthy tissues to allow primary tumor cells to escape their primary environment and subsequently invade distant tissues to form distant metastases.
Interestingly, protein matrices such as extracellular matrix and fibrin clots are typically dynamic, that is the matrix can be formed and degraded as part of pathological processes or normal physiological processes. Indeed, hemostasis can be viewed as the maintenance of an equilibrium between the formation of fibrin clots and the proteolytic degradation of those clots (fibrinolysis) by enzymes or coagulation factors including plasmin.
Methods for detecting or monitoring the formation of protein matrices such as blood clots (thrombi) include for example methods of determining blood coagulation e.g., prothrombin time, thrombin clotting time or the Clauss method for fibrinogen testing, and there are commercial devices that can perform such coagulation testing in portable, point-of-care formats. However, tests for detecting or monitoring fibrinolysis are generally limited to complex methodologies in specialized laboratories and on native fibrin clots, such as, for example thromboelastometry (TEM), and are not applicable to point of care or field diagnostics. Alternative approaches employ colorimetric or fluorescent detection methodologies and are, too, expensive, complex and furthermore negatively affected by the color or turbidity of a sample, and hence ineffective for timely and sensitive detection and monitoring of fibrinolysis.
There therefore exists a need for new and improved apparatus and methods for detecting and/or monitoring proteolysis of protein matrices. In particular, for example, electrochemical and related analytical technology which detect and monitor proteolysis activities such as fibrinolysis or the breakdown of collagen, extracellular matrix, or other similar protein matrices, may provide valuable diagnostics and monitoring tools for medicine and wellness.